Amino Acid and Peptides Biochemistry PDF

AMINO ACID AND PEPTIDES **Biomedical Importance:** - **L-α-amino Acids**: These are the building blocks of proteins and help in many body functions like nerve signaling and making essential compounds. - **Peptides**: Short chains of amino acids that act as hormones or messengers i...

AMINO ACID AND PEPTIDES **Biomedical Importance:** - **L-α-amino Acids**: These are the building blocks of proteins and help in many body functions like nerve signaling and making essential compounds. - **Peptides**: Short chains of amino acids that act as hormones or messengers in the body. - **Microbial Peptides**: Some are useful, like antibiotics, but others can be toxic and cause health problems. - **Essential Amino Acids**: Humans need to get 10 of these from their diet because the body can't make them in enough amounts. **Properties of Amino Acids:** - **Genetic Code**: Only 20 types of amino acids are used to build proteins, though there are over 300 types found in nature. - **Modifications**: Amino acids can be changed after they are added to proteins, affecting how proteins work. - **Chirality**: Most amino acids are chiral (have a specific orientation), and proteins only use the L-form. - **D-Amino Acids**: Some natural amino acids are in the D-form and are found in certain bacteria and peptides. **Charges of Amino Acids:** - **Ionizable Groups**: Amino acids have carboxyl (-COOH) and amino (-NH3+) groups that can gain or lose protons. - **Carboxyl Group**: Acts as a weak acid and is stronger than the amino group. At physiological pH (7.4), it mostly exists as -COO-. - **Amino Group**: Acts as a weak base and mostly exists as -NH3+ at physiological pH. - **Zwitterions**: Amino acids often exist with no net charge due to equal positive and negative charges in solution. **pKa Values:** - **pKa**: Measures the strength of a weak acid. Lower pKa means stronger acid. - **Histidine and Arginine**: Have special groups with positive charges that affect their pKa values and behavior in solution. **Isoelectric Point (pI):** - **pI**: The pH at which an amino acid has no net charge. It is the average of the pKa values surrounding the neutral form. - **Separation**: pI helps in separating amino acids and proteins in lab techniques like electrophoresis and chromatography, based on their charge at different pH levels. **pKa Values and Environment:** - **pKa Variation**: The pKa of amino acids changes based on their environment. - **Polar vs. Nonpolar**: Polar environments favor charged forms (e.g., -COO- or -NH3+), while nonpolar environments favor uncharged forms (e.g., -COOH or -NH2). - **Protein Influence**: pKa values in proteins can vary significantly due to their environment. For example, the pKa of an aspartic acid in a protein can shift by over six pH units. **Solubility and Melting Points:** - **Solubility**: Amino acids are soluble in polar solvents (e.g., water) but insoluble in nonpolar solvents (e.g., benzene). - **Melting Points**: Amino acids have high melting points (\>200°C) due to their ionic character. - **UV Absorption**: Amino acids are colorless, but tyrosine, phenylalanine, and tryptophan absorb UV light, with tryptophan contributing most to UV absorption at 280 nm. **Properties Determined by -R Groups:** - **Glycine**: Fits into tight spaces in proteins due to its small size. - **Hydrophobic Groups**: Amino acids like alanine and leucine are usually found inside proteins. - **Charged Groups**: Basic and acidic amino acids stabilize protein structures through ionic interactions and play roles in enzyme reactions. - **Histidine**: Can act as both an acid and a base in enzymatic reactions. - **Nucleophiles**: Serine and cysteine have reactive groups involved in enzymatic catalysis, while threonine does not. **Chemical Reactions:** - **Functional Groups**: Carboxylic acid groups form esters and amides, amino groups undergo acylation and amidation, and -OH and -SH groups participate in oxidation. - **Peptide Bond Formation**: The most crucial reaction for amino acids is forming peptide bonds, which link them into proteins. **Primary Structure of Proteins:** - **Sequence**: The primary structure of a protein is the sequence of amino acids in its polypeptide chain. - **Naming Peptides**: Amino acids in peptides are named with a \"-yl\" suffix, and peptides are named based on the sequence of these residues. For example, Lys-Leu-Tyr-Gln is called lysyl-leucyl-tyrosyl-glutamine. **Peptide Notation:** - **Prefixes**: \"Tri-\" and \"octa-\" refer to the number of amino acid residues, not peptide bonds. - **Drawing Peptides**: Use a zigzag for the backbone with atoms in a repeating order: α-nitrogen, α-carbon, carbonyl carbon. Add hydrogen atoms to α-carbons and peptide nitrogens, and oxygen to carbonyl carbons. Use three-letter abbreviations connected by lines for clarity. **Unusual Amino Acids:** - **Non-Standard Amino Acids**: Peptides can contain non-protein amino acids or atypical bonds. Examples include glutathione with a non-α peptide bond and TRH with cyclized glutamate. - **Peptide Examples**: Some antibiotics and peptides from lower organisms contain unusual amino acids. **Peptide Properties:** - **Polyelectrolytes**: Peptides are charged due to their terminal groups and any acidic/basic R groups, depending on pH and pKa values. - **Peptide Bond**: Exhibits partial double-bond character, which restricts rotation and makes the bond rigid, affecting protein structure. **Conformation:** - **Peptide Folding**: Folding occurs during peptide synthesis and depends on the amino acid sequence and noncovalent interactions. Common structures include α-helices and β-pleated sheets. **Amino Acid Analysis:** - **Hydrolysis**: Peptide bonds are broken using hot HCl to release free amino acids. - **Detection**: Amino acids are then reacted with specific reagents (e.g., 6-amino-N-hydroxysuccinimidyl carbamate or ninhydrin) and analyzed by high-pressure liquid chromatography or colorimetric methods. PROTEINS: DETERMINATION OF PRIMARY STRUCTURE **Biomedical Importance of Proteins** **Critical Roles:** - **Cytoskeleton**: Maintains cellular shape and integrity. - **Muscle**: Actin and myosin enable contraction. - **Transport**: Hemoglobin carries oxygen. - **Immune Response**: Antibodies identify and neutralize foreign invaders. - **Enzymes**: Catalyze reactions for energy, biomolecule synthesis/degradation, and gene processing. - **Receptors**: Detect hormones and environmental signals. **Protein Purification Techniques:** 1. **General Purification**: - **Classical Methods**: Exploit solubility differences (e.g., isoelectric precipitation, salting out). - **Chromatographic Techniques**: - **Column Chromatography**: Separates based on charge, hydrophobicity, or ligand-binding using a column with beads. - **Size Exclusion Chromatography**: Separates by Stokes radius, where larger molecules exit the column first. - **Ion Exchange Chromatography**: Separates based on charge interactions with positively or negatively charged beads. - **Hydrophobic Interaction Chromatography**: Uses hydrophobic beads to bind proteins with hydrophobic surfaces. - **Affinity Chromatography**: Exploits specific interactions between proteins and their ligands. 2. **Advanced Techniques**: - **High-Pressure Liquid Chromatography (HPLC)**: Utilizes high pressure and non-compressible materials for resolving complex mixtures. - **Reversed-Phase HPLC**: Uses hydrophobic stationary phases and gradients of organic solvents. **Protein Purity Assessment:** 1. **SDS-PAGE**: Uses sodium dodecyl sulfate (SDS) to denature proteins, allowing separation based on size. Proteins are stained for visualization. 2. **Isoelectric Focusing (IEF)**: Separates proteins based on their isoelectric point (pI) in a pH gradient. Often combined with SDS-PAGE for two-dimensional electrophoresis, which separates proteins based on both pI and molecular weight. **Historical Insight:** - **Fredrick Sanger**: First to determine the sequence of a polypeptide, starting with insulin. His methods included reducing disulfide bonds and sequencing peptides. **Visual Representations** - **Figure 4--1**: Liquid chromatography apparatus components. - **Figure 4--2**: Size-exclusion chromatography showing large vs. small molecules. - **Figure 4--3**: Methods for cleaving disulfide bonds. - **Figure 4--4**: SDS-PAGE used for protein purification and visualization. - **Figure 4--5**: Two-dimensional electrophoresis combining IEF and SDS-PAGE. **Two-Dimensional IEF-SDS-PAGE** **Principle**: - **Isoelectric Focusing (IEF)**: Proteins are separated in the first dimension based on their isoelectric point (pI), which is the pH at which a protein has no net charge. Proteins migrate through a pH gradient until they reach the point where their net charge is zero. - **SDS-PAGE**: The second dimension separates proteins based on size. After IEF, the gel is laid horizontally on an SDS-PAGE gel, where proteins are further resolved by their molecular weight. **Advantage**: - This method provides high-resolution separation of proteins compared to conventional SDS-PAGE, allowing for better visualization and analysis of individual protein components. **Sanger's Method** **Overview**: - Developed by Frederick Sanger, this method labels the amino-terminal residue of a peptide with 1-fluoro-2,4-dinitrobenzene (Sanger's reagent). **Procedure**: 1. **Labeling**: Peptides are reacted with Sanger's reagent. The reagent binds to the free α-amino group at the amino-terminal end of the peptide. 2. **Identification**: The amino acid content is determined based on the amount of reagent that reacts. Amino-terminal lysines are identified as they react with two molecules of the reagent. 3. **Sequencing**: Working backwards from the labeled amino acids allows for the determination of the full peptide sequence. **Significance**: - Sanger's method was crucial in determining the amino acid sequences of proteins, such as insulin, which earned Sanger a Nobel Prize. **Edman Degradation** **Overview**: - Developed by Pehr Edman, this method sequentially identifies the amino-terminal residue of peptides using phenylisothiocyanate (Edman's reagent). **Procedure**: 1. **Reaction**: Phenylisothiocyanate reacts with the amino-terminal residue to form a phenylthiohydantoin (PTH) derivative. 2. **Cleavage**: The PTH derivative is removed under mild conditions, leaving a new amino-terminal residue on the peptide. 3. **Identification**: The PTH derivative is analyzed by chromatography to identify the amino acid. This process is repeated to determine the sequence of amino acids in the peptide. **Advantage**: - Allows for the sequencing of multiple residues from a single peptide sample. Automated Edman sequencers can analyze picomole amounts of peptide. **Peptide Cleavage** **Purpose**: - Large polypeptides are cleaved into smaller peptides for easier sequencing. Cleavage can also be necessary to overcome blocking groups or posttranslational modifications. **Methods**: 1. **Chemical Cleavage**: - **Cyanogen Bromide (CNBr)**: Cleaves at methionine residues. - **Hydroxylamine**: Cleaves asparagine-glycine bonds. - **o-Iodosobenzene**: Cleaves at tryptophan residues. - **Mild Acid**: Cleaves aspartic acid-proline bonds. 1. **Enzymatic Cleavage**: - **Trypsin**: Cleaves after lysine and arginine residues. - **Chymotrypsin**: Cleaves after hydrophobic amino acids. - **Endoproteinase Lys-C**: Cleaves after lysine residues. - **Endoproteinase Arg-C**: Cleaves after arginine residues. - **V8 Protease**: Cleaves after glutamic acid residues, particularly if followed by hydrophobic residues. **Post-Cleavage**: - Peptides are purified and analyzed by techniques like reversed-phase HPLC or SDS-PAGE before sequencing. **Mass Spectrometry** **Principle**: - Measures the mass-to-charge ratio of ions to determine molecular weights and identify molecules. **Process**: 1. **Ionization**: Molecules are ionized (typically protonated) and introduced into the mass spectrometer. 2. **Acceleration**: Ions are accelerated through an electric field. 3. **Deflection**: Ions pass through a magnetic field, which deflects them based on their mass. 4. **Detection**: The deflected ions strike a detector, and the amount of current generated is used to determine their mass. **Types**: - **Time-of-Flight (TOF) Mass Spectrometry**: Measures the time ions take to reach the detector, which is inversely proportional to their mass. - **Matrix-Assisted Laser Desorption/Ionization (MALDI)**: Allows for the analysis of large molecules by embedding them in a matrix that absorbs laser energy. - **Electrospray Ionization (ESI)**: Directly introduces peptides from a liquid phase into the mass spectrometer. **Tandem Mass Spectrometry (MS/MS)**: - Uses two mass spectrometers in series. The first spectrometer isolates a peptide, and the second breaks it into fragments (collision-induced dissociation). The fragment masses help determine the peptide's sequence. **Genomics and Proteomics** **Genomics**: - Involves sequencing entire genomes to infer protein sequences from DNA. This has greatly accelerated the ability to predict protein structures from genetic information. **Proteomics**: - The study of the entire set of proteins expressed by a cell, tissue, or organism at a specific time. It includes: - **Two-Dimensional Electrophoresis**: Separates proteins by both isoelectric point and size. - **Gene Arrays (DNA Chips)**: Measure mRNA expression levels, which can indicate which proteins are being synthesized. **Bioinformatics**: - Uses computational tools to predict protein functions based on sequence similarity to known proteins. It involves comparing sequences to identify structural and functional domains. PROTEINS: HIGH ORDERS OF STRUCTURE **Biomedical Importance of Protein Structure** Proteins are fundamental to numerous biological functions and their diverse structures reflect their wide range of roles: - **Catalysis**: Proteins (enzymes) accelerate metabolic reactions, enabling essential processes like digestion, cellular respiration, and biosynthesis. - **Structural Integrity**: Proteins provide structural support to cells and tissues, forming critical components like hair, bones, tendons, and teeth. - **Cellular Motion**: Motor proteins, such as myosin and dynein, facilitate movement within cells and the movement of cells themselves. **Form and Function**: - The structure of a protein determines its function. For example, the unique folding and three-dimensional arrangement of enzymes create specific active sites for substrate binding and catalysis. **Maturation and Posttranslational Modifications**: - Proteins must fold correctly to achieve their functional form. Posttranslational modifications, such as the addition of chemical groups or the removal of peptide segments, are crucial for the proper function of many proteins. - **Genetic or nutritional deficiencies** that disrupt protein maturation can lead to diseases. Examples include: - **Creutzfeldt-Jakob Disease** and **Scrapie**: Misfolded proteins (prions) cause neurodegenerative diseases. - **Alzheimer's Disease**: Characterized by abnormal protein aggregates (amyloid plaques). - **Bovine Spongiform Encephalopathy**: Also known as \"mad cow disease,\" caused by prion proteins. - **Scurvy**: Results from vitamin C deficiency, which impairs collagen synthesis and maturation. **Conformation vs. Configuration** **Configuration**: - Refers to the fixed spatial arrangement of atoms that requires breaking and reforming covalent bonds to change. For example, the difference between L- and D-amino acids. **Conformation**: - Refers to the three-dimensional shape of a molecule, which can change without breaking covalent bonds, typically through rotation around single bonds. **Protein Classification** **Initial Classification**: - Early methods categorized proteins based on properties such as solubility and shape: - **Soluble Proteins**: Extractable in physiological conditions. - **Membrane Proteins**: Require detergents for extraction. - **Globular Proteins**: Compact, spherical, or ovoid, often enzymes. - **Fibrous Proteins**: Extended structures, like collagen and keratin, providing structural support. **Modern Classification**: - Advances in sequencing and structural biology have led to classifications based on amino acid sequences and structural similarities. Terms from early classifications are still commonly used. **Modular Nature of Protein Structure** **Modular Principles**: - Proteins are built from a set of amino acids linked by peptide bonds. The folding and function are guided by modular building blocks and interactions. - Proteins can adopt an enormous number of conformations (e.g., 10\^50), but they fold into specific functional forms due to their modular nature. **The Four Orders of Protein Structure** 1. **Primary Structure**: - The linear sequence of amino acids in a polypeptide chain. 2. **Secondary Structure**: - Local folding patterns within a polypeptide chain, including: - **Alpha Helix**: A right-handed coil with hydrogen bonds stabilizing the structure. Each turn of the helix includes about 3.6 residues. - **Beta Sheet**: Formed by extended strands connected laterally by hydrogen bonds, aligning side by side. 3. **Tertiary Structure**: - The overall three-dimensional shape of a single polypeptide chain, formed by the folding of secondary structures into a compact, functional unit. 4. **Quaternary Structure**: - The arrangement of multiple polypeptide chains (subunits) into a single functional protein complex. **Secondary Structure Details** **Alpha Helix**: - **Phi (Φ) and Psi (Ψ) Angles**: The backbone angles that define the helix's conformation. - **Stability**: Hydrogen bonds between the carbonyl oxygen and the amide hydrogen of the fourth residue down the chain stabilize the helix. Proline can disrupt helices due to its unique structure, and glycine often causes bends. **Beta Sheet**: - **Orientation**: Beta sheets consist of beta strands linked by hydrogen bonds. Strands can be parallel or antiparallel. - **Stability**: Stabilized by hydrogen bonding between the backbone amide and carbonyl groups of adjacent strands. **Ramachandran Plot**: - A graphical representation of allowed phi and psi angles for protein backbone conformations. It helps visualize which angles are sterically allowed or prohibited. **The Beta Sheet** **Overview:** - **Structure:** The β sheet is a secondary structure in proteins where amino acid residues form a zigzag or pleated pattern when viewed edge-on. The peptide backbone is highly extended compared to the α helix. - **Stability:** Like the α helix, β sheets are stabilized by hydrogen bonds. However, these bonds occur between different segments of the β sheet, not within the same segment. **Types of β Sheets:** 1. **Parallel β Sheet:** Adjacent segments run in the same direction (amino to carboxyl). Hydrogen bonds are slanted. 2. **Antiparallel β Sheet:** Adjacent segments run in opposite directions. Hydrogen bonds are more perpendicular to the backbone and are more uniformly spaced. **Characteristics:** - **Orientation:** β sheets often exhibit a right-handed twist. - **Representation:** Schematic diagrams represent β sheets as arrows pointing from amino to carboxyl terminal directions. **Loops & Bends:** **Function:** - **Loops and Bends:** These structures join different secondary structures like α helices and β sheets. They are crucial for protein function and can be involved in substrate binding and protein interactions. **β Turn:** - **Structure:** Involves four amino acids with the first residue hydrogen-bonded to the fourth, creating a tight 180-degree turn. Commonly includes proline and glycine. **Loops:** - **Role:** They bridge secondary structure elements and can be irregular but essential for protein function. They may form part of epitopes for antibody binding. **Tertiary & Quaternary Structure** **Tertiary Structure:** - **Definition:** The overall three-dimensional shape of a single polypeptide chain, including how secondary structures (helices, sheets) come together. - **Domains:** Functional units within proteins that perform specific tasks, such as substrate binding. **Quaternary Structure:** - **Definition:** The arrangement of multiple polypeptide chains (subunits) in a protein. Can be monomeric (one chain) or oligomeric (multiple chains). - **Types:** - **Homodimers:** Two identical polypeptide chains. - **Heterodimers:** Two different polypeptide chains. - **Homotetramers:** Four identical chains. **Stabilizing Factors:** - **Noncovalent Interactions:** Hydrophobic interactions, hydrogen bonds, and salt bridges contribute to protein stability. - **Covalent Bonds:** Disulfide bonds between cysteine residues can further stabilize protein structures. **Visualization:** - **Ribbon Diagrams:** Illustrate the conformation of the polypeptide backbone with cylinders and arrows for helices and sheets. - **Simplified Models:** Line diagrams showing the path of the backbone, often with key side chains highlighted for structural-functional relationships. **Three-Dimensional Structure of Proteins** **X-Ray Crystallography**: - This method determines protein structures by crystallizing the protein and analyzing how it diffracts X-rays. - Proteins must form ordered crystals, which is achieved by adjusting conditions like pH and using precipitating agents. - Despite advances in computing, crystallization remains a major challenge. - The technique has provided detailed structures for thousands of proteins, and most crystallized proteins reflect their solution forms. **Nuclear Magnetic Resonance (NMR) Spectroscopy**: - NMR spectroscopy measures how nuclei absorb radio frequency energy, giving insight into protein structures in solution. - It is useful for observing changes in protein conformations, such as during ligand binding. - Current limitations restrict this technique to relatively small proteins (≤ 20 kDa). **Molecular Modeling**: - Computer models can predict protein structures based on known data or simulate changes in conformation. - These models can be used to explore how proteins fold or react to environmental changes. **Protein Folding** **Native Conformation**: - Proteins naturally fold into the most thermodynamically stable conformation. - This process is guided by the primary sequence of the protein. **Folding Process**: - Initial folding occurs into secondary structures (α-helices and β-sheets). - Hydrophobic regions drive the formation of a molten globule, which rearranges into the mature structure. - Folding is modular, with each structural element aiding in achieving the final conformation. **Auxiliary Proteins**: - Chaperones like hsp70 and hsp60 assist in proper folding by preventing aggregation and helping proteins reach their native states. - Protein disulfide isomerase helps in forming stable disulfide bonds, while proline-cis,trans-isomerase facilitates the correct configuration of proline residues. **Neurologic Diseases from Altered Protein Conformation** **Prions**: - Prion diseases (e.g., Creutzfeldt-Jakob disease, mad cow disease) involve proteins that misfold and induce misfolding in normal proteins. - These misfolded proteins form insoluble aggregates and are transmitted without DNA or RNA. **Alzheimer's Disease**: - Characterized by the aggregation of β-amyloid protein into plaques, leading to neurodegeneration. - The misfolding of β-amyloid from a soluble to an aggregated form is central to disease progression. **Collagen and Posttranslational Processing** **Collagen**: - Collagen, a fibrous protein, provides structural support in connective tissues and is synthesized as a precursor called procollagen. - The collagen triple helix consists of three polypeptide chains wound around each other, stabilized by hydrogen bonds and covalent cross-links. **Posttranslational Modifications**: - Procollagen undergoes modifications like hydroxylation and glycosylation. - Proteolytic cleavage and covalent cross-linking are essential for forming mature collagen fibers. **Disorders**: - Scurvy (vitamin C deficiency) and Menkes' syndrome (copper deficiency) impact collagen maturation. - Genetic disorders such as osteogenesis imperfecta and Ehlers-Dahlos syndrome involve defects in collagen biosynthesis, affecting connective tissue strength and flexibility.